In order to quantify the hormone present in an assay tube, it is necessary to determine the amount of hormone bound to the antibody. A radioisotopically labelled steroid is used for this purpose and according to Niwender, G. D., Akbar, A. M., and Nett, J. M. in "Methods in Enzymology, Vol. 36, Hormone Action, Part A: Steroid Hormones", Academic Press, 1975, at pp. 16-33, it is not possible to directly radioiodinate the cyclopentanophenanthrene nucleus or its substituents. Furthermore, these authors report that, although phenolic steroids can be radioiodinated directly at the 2 and 4 positions, this seems to alter the configuration of the molecule and affects the binding to the antibody because the physical dimensions of an iodine atom approximate those of the complete phenolic A ring in the molecule.
By conjugating the steroid to a tyrosene-containing protein, for example to bovine serum albumin (BSA), it is possible to radioiodinate the protein without affecting the ability of the steroid to bind to an antibody. However, there is a distinct difference between the tyrosene-containing protein and the procedure of this invention in that herein the steroid is directly iodinated. Furthermore, the use of tyrosene-containing protein does not insure that only one steroid molecule is attached to each albumin molecule and, in fact, the steroid-to-protein ratio is at least 20:1.
To prepare one steroid per radioiodinated protein, Niwender et al uses the methyl ester of tyrosine (TME) conjugated steroid protein derivative. The phenolic tyrosine ring of this derivative is easily iodinated with Na.sup.125 I by Chloramine T procedures of iodination of proteins. The steroid-protein-TME-.sup.125 I thus prepared is shown to possess antigenic specificity to steroid antiserum and could be used for the radioimmunoassay procedures of estrogen.
According to G. E. Abraham and W. D. Odell, "Solid Phase Radioimmunoassay of Serum Estradiol-17 beta" in "Immunologic Methods in Steroid Determinations." Peron and Caldwell (1970), estrogen (E.sub.2) may be labeled with radioisotopes emitting beta- or gamma-radiations using radioactive halogens such as .sup.82 Br, .sup.125 I and .sup.131 I at positions C-2 and C-4 in the phenolic ring. However, the short half-life of the halogens limits its use to a few days only. Another factor, these authors report, that almost rules out the radio-active halogens as a possible marker on the E.sub.2 molecule is inherent in their physical properties, namely their highly electronegative characteristics which affect the net charge of the molecules and the radioactive halogens with the longest half-life are relatively large atoms that would interfere with the steric fitness of the antibody active cites. According to these authors, directly iodinated estradiol 17-beta at positions 2 and 4 is unable to bind the anti-serum because of the interference induced by the relatively large atoms of iodine with the steric fitness of these active cites of the antibody.
It is known that the steroid synthesizing organs such as the gonads, the fetoplacental unit and the adrenal cortex utilize the same precursor substances for the synthesis of steroid hormones. Also, steroid specific receptor proteins are present in various normal tissues and in certain malignant tumors. Receptor cites for both estrogen and progesterone are usually found in hormone dependent tumors such as human breast cancer.
When tritiated estradiol is injected to patients with breast cancer, those patients responding to endocrine treatment usually concentrate more tritiated hormones in their tumor tissue than those patients who do not respond to hormonal treatment. The uptake of tritiated hormone by the tumor tissue is facilitated by the presence of specific estrogen receptor sites in such tumors. Other tumors such as endometrial carcinoma and prostate cancer are known to possess specific receptor sites for steroid hormones like estrogen, progesterone and testosterone.